The present invention relates to methods for the transformation and regeneration of transformed embryogenic tissue of coniferous plants. In particular, the invention relates to improved methods for transforming embryogenic tissue of coniferous plants and for regenerating transformed embryogenic tissue of coniferous plants. The invention is well suited to the transformation and regeneration of transformed embryogenic tissue of plants of the subgenus Pinus of pines.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended Bibliography.
Reforestation, the controlled regeneration of forests, has become an integral part of forest management in order to secure a renewable and sustainable source of raw material for production of paper and other wood-related products. Forest trees can be regenerated by either sexual or asexual propagation. Sexual reproduction of seedlings for reforestation has traditionally been the most important means of propagation, especially with coniferous species.
Tree improvement programs with economically important conifers (e.g., Pinus, Picea, and Pseudotsuga species) have applied genetic principles of selection and breeding to achieve genetic gain. Based on the results of progeny tests, superior maternal trees are selected and used in “seed orchards” for mass production of genetically improved seed. The genetic gain in such an open-pollinated sexual propagation strategy is, however, limited by the breeder's inability to control the paternal parent. Further gains can be achieved by control-pollination of the maternal tree with pollen from individual trees whose progeny have also demonstrated superior growth characteristics. Yet sexual propagation results in a “family” of seeds comprised of many different genetic combinations (known as siblings), even though both parents of each sibling seed are the same. As not all genotype combinations are favorable, the potential genetic gain is reduced due to this genetic variation among sibling seeds.
In addition to these genetic limitations, large-scale production of control pollinated seeds is expensive. These economic and biological limitations on large-scale seed production have caused considerable interest to develop in the industry for applying asexual methods to propagate economically important conifers.
The use of asexual propagation permits one to apply what is known as a very high selection intensity (that is, to propagate only progeny showing a very high genetic gain potential). These highly desirable progeny have unique genetic combinations that result in superior growth and performance characteristics. Thus, with asexual propagation it is possible to multiply genetically select individuals while avoiding a concomitant reduction of genetic gain due to within-family variation. Asexual propagation of trees can be accomplished by methods of grafting, vegetative propagation, and micropropagation. Micropropagation by somatic embryogenesis refers to methods whereby embryos are produced in vitro from small pieces of plant tissue or individual cells. The embryos are referred to as somatic because they are derived from the somatic (vegetative) tissue, rather than from the sexual process. Both vegetative propagation and micropropagation have the potential to capture all genetic gain of highly desirable genotypes. However, unlike conventional vegetative propagation methods, somatic embryogenesis is amenable to automation and mechanization, making it highly desirable for large-scale production of planting stock for reforestation. In addition, somatic embryogenic cultures can easily be preserved in liquid nitrogen. Having a long-term cryogenic preservation system offers immense advantages over other vegetative propagation systems which attempt to maintain the juvenility of stock plants.
One source of new genetic material for use in reforestation or tree improvement programs is plant tissue that has been transformed to contain one or more genes of interest. Genetic modification techniques enable one to insert exogenous nucleotide sequences into an organism's genome. A number of methods have been described for the genetic modification of plants, including transformation via biolistics and Agrobacterium tumefaciens. All of these methods are based on introducing a foreign DNA into the plant cell, isolation of those cells containing the foreign DNA integrated into the genome, followed by subsequent regeneration of a whole plant.
A significant problem in production of transgenic plants is how to recover only transformed cells following transformation, while causing minimal perturbations to their health so that they can proliferate, give rise to differentiating cultures and ultimately regenerate transgenic plants.
It is well known that embryogenic cultures, in general, and pine embryogenic cultures, specifically, can experience significant decline in regeneration potential under stressful culture conditions. Stresses to the cells during and after transformation can include the perturbations of the transformation process (which may include co-cultivation with Agrobacteria, bombardment with microprojectiles, chemical treatments, electroporation or mechanical shearing), any measures that allow preferential growth of transformed cells while selectively killing or depressing the growth or regeneration of untransformed cells (referred to as “selection”), exudates released from dying cells in the culture, and/or the elicitation of transgene activity in the transformed cells (for “positive selection” or detection of the activity of “visual marker genes”). It stands to reason that when transformed cells are not maintained in sufficient health to allow their survival through these stresses, not only will they fail to give rise to transgenic plants, they may never be detected as transformed in the first place.
Regeneration of transformed plants from transformed cultures of pine has been difficult. Reports of pine transformation and regeneration include the following:
U.S. Pat. No. 4,459,355 (Cello and Olsen, 1984) describes a method for using Agrobacterium tumefaciens to transform plant cells. The patent claims transformation of any dicotyledon or any gymnosperm (e.g. loblolly pine, cedar, Douglas fir). However, no example of transformation of any gymnosperm is given. Thus, a claim of stable transformation of pines following inoculation with Agrobacterium tumefaciens was allowed in U.S. Pat. No. 4,886,937 (Sederoff et al., 1989).
U.S. Pat. No. 4,886,937 also claims the transformed pine obtained from inoculation with Agrobacterium tumefaciens. However, no transformed pine plants were obtained in the examples, which are restricted to formation of non-regenerable galls following inoculation of seedlings. Further work by researchers in the same lab, using Agrobacterium tumefaciens to inoculate pine and spruce somatic embryogenic cultures, was published (Wenck et al., 1999). In the work described in that publication, stable transformation of both species was achieved, but while plants were regenerated from the transformed spruce cultures, no plants could be obtained from the loblolly pine cultures.
In particle-mediated gene transfer, the DNA of interest is precipitated onto the surface of carrier particles which are subsequently accelerated toward a piece of target tissue. The carrier particles penetrate the cell wall of the plant cell, wherein the DNA can be expressed, and may integrate with the chromosomal DNA. In some instances stable expression results if the transforming DNA integrates with the chromosomal DNA (Walter et al. 1994), but sorbitol pre-treatments described as important for obtaining stable expression were not taught for regeneration of transformed pine plants (Walter et al. 1997), perhaps because, as we found, such treatments can also be detrimental to the regeneration of pine plants. To obtain high frequency gene transfer and regeneration of plants in the genus Pinus, we developed a variety of high gelling agent or high osmoticum preparation media for use before transformation and selection in pines, described in U.S. patent application Ser. No. 09/318,136 filed on 25 May 1999 and New Zealand Patent No. 336149, each incorporated herein by reference.
Although regeneration of planting stock of transformed pine via biolistic processes has been reported as described above, transformed sublines and transformed plants had never been detected or recovered from pine embryogenic lines of certain genetic backgrounds. One problem has been that embryogenic masses from many species of pines cannot be maintained for long periods on media before culture decline is observed in many lines. For example, culture decline is observed to occur frequently with progeny of the P. taeda elite selection 7-56, an unfortunate circumstance because these crosses are considered genetically valuable and are used in many breeding programs. Although such material would be a desirable substrate for transformation, any delay in embryo formation, which can be caused for example by the sometimes lengthy period of selection following transformation, and the period of bacterial eradication following particularly Agrobacterium transformation, exacerbates the problem of culture decline.
A measure taken to speed up selection and increase proliferative health followed the observation that abscisic acid (ABA) in the gelled media is important in order to obtain transformed embryogenic masses from certain embryogenic lines, while it does not prevent growth of stably transformed embryogenic masses of many other pine genotypes, including interspecific hybrids. In other words, the addition of ABA to the media used for transformation and post-transformation recovery and growth is either neutral, or beneficial for certain genotypes. Because maintenance, recovery and selection media containing ABA support as good or better growth rates as media lacking ABA, selection of transformed lines is accomplished more rapidly, increasing the health of the cells going into the embryo development phase and decreasing the time prior to differentiation of embryos. Thus, regeneration of transformed plants is enhanced as a result of increased proliferative health of transformed tissue by the inclusion of ABA in the culture media. It has also been found that the presence of ABA in the preparation media for transformation, i.e., the preparation media used for bombardment or co-cultivation with Agrobacterium, can in some genotypes assist transformed cells to survive the stress of transformation.
The importance of abscisic acid (ABA) during the development and maturation of zygotic embryos is well known, and ABA has been used routinely to stimulate terminal embryo development in somatic embryogenic systems (von Arnold and Hakman, 1988). For example, U.S. Pat. No. 4,957,866 teaches the use of ABA in the terminal embryo development media. Likewise, in U.S. Pat. Nos. 5,034,326 and 5,036,007 the phytohormone ABA along with activated carbon has been reported to be beneficial in gelled embryo development media for various conifers. U.S. Pat. No. 5,294,549 teaches the incorporation of ABA and gibberellic acid into the embryo development media. U.S. Pat. Nos. 5,187,092, 5,183,757, and 5,236,841 teach the use of ABA in the terminal embryo development step in conifer somatic embryogenesis. In each of these methods ABA is added for the purpose of facilitating terminal embryo development to the cotyledonary stage for the regeneration of plants.
Terminal development of embryos for the regeneration of plants from somatic embryogenic tissue is effected not only by the addition of ABA but also by affecting the water potential of the embryogenic tissue, either by the use of polyethylene glycol or other osmotica (see for example U.S. Pat. No. 5,036,007) or by separating the somatic embryos from a liquid medium by a porous support, or by introducing a gelling agent (e.g. gellan gum) into the medium in larger than normal quantities (see for example in U.S. Pat. No. 6,200,809) for the purposes of obtaining terminal embryo development.
Heretofore there has been no evidence that the use of ABA or manipulation of the water potential during selection, either in plants in general or with coniferous species, would be beneficial. In fact, although it is well known that these factors are important in the terminal development of embryos both in vivo and in vitro, their ability to stimulate recovery of transformed embryogenic tissue so that proliferative growth can resume in transformed cells of elite lines of P. taeda and hybrids was unexpected.
The developmental stage of the explant tissue used to initiate embryogenic cultures in conifers is critically important. Pines have proven much more restricted than spruces in terms of the responsive embryo development stage for somatic embryogenic culture initiation. To be successful in pines, one must use only very immature embryos (or seeds containing such immature embryos). The size of the developing embryo, usually measured as length, has frequently been used to determine the appropriate developmental stage for culture initiation in many plant species. This has been the case with loblolly pine where it was found that the embryogenic culture initiation occurred most frequently when the dominant zygotic embryo was less than about 0.5 mm in length.
Because it is difficult to measure the size of very immature differentiated embryos, embryo staging systems have also been used to make the determination of the appropriate developmental stage easier. These staging systems are based on several factors, including various morphological characteristics of the embryo. An embryo staging system proposed by Hakman and von Arnold (1988), which is commonly utilized in the industry, has the following three distinct stages. Stage 1 embryos are small differentiated embryos consisting of an embryonic region of small, densely cytoplasmic region subtended by a suspensor comprised of long, highly vacuolated cells. Stage 2 embryos are further differentiated embryos with a prominent embryonic region that becomes more opaque and assumes a smooth and glossy surface. Stage 3 embryos are further differentiated embryos which show visible cotyledonary primordia. Thus, stage 1 and 2 embryos are at a pre-cotyledonary stage of development, while stage 3 embryos are cotyledonary. As used herein, the term “pre-stage 3 embryo” means a differentiated pre-cotyledonary embryo (i.e., a stage 1 or stage 2 embryo). Although the above three-stage system was first used with somatic embryos of spruce, it is generally applicable to both somatic and zygotic embryos of all conifer species.
As described in U.S. patent application Ser. No. 09/318,136 filed on 25 May 1999 and New Zealand Patent No. 336149, each incorporated herein by reference, it has been observed that the presence in the tissue of embryos at the proper precotyledonary stage was both necessary and sufficient for efficient transformation of the genus Pinus. Differentiation of tissue to the appropriate stage of embryo development was aided by manipulation of osmoticum and gelling agent concentrations to obtain matrix potentials sufficient to prepare the tissue for transformation. It was further observed that transfer of precotyledonary embryos to a maintenance medium, with or without a selection agent, allowed cells on the embryos to re-initiate secondary somatic embryogenesis, and the embryogenic tissue so derived is then able to resume proliferative growth. Following transformation, selection of such embryogenic tissue is needed in order to generate transgenic embryogenic cell lines.
It had previously been found that both ABA and manipulation of gelling agent concentrations can contribute to more efficient culture initiation in pine. U.S. Pat. No. 5,506,136 by Becwar et al. (1996) describes the use of a reduced gelling agent concentration to obtain higher frequency of initiation. U.S. Pat. No. 5,856,191 by Handley (1999) employs ABA as an improvement upon the methods described in U.S. Pat. No. 5,506,136 in both the initiation and maintenance medium for pine embryogenic cultures prior to cryopreservation. The utility of ABA in obtaining improved conditions for culture initiation was unexpected, as in this case.
In U.S. Pat. No. 5,856,191, the use of ABA is coupled with another method that is known to regulate conifer embryo development, namely manipulation of the matrix potential of the gelled medium.
Accordingly, we investigated whether the addition of ABA or the manipulation of the matrix potential of the gelled medium might be able to stimulate recovery of transgenic cell lines from selection, in a mode of action similar to the stimulation that these agents are able to provide, separately or together, in initiation of primary somatic embryogenesis in the genus Pinus. 
Thus, it is an object of the present invention to provide an improved method for the selection of transformed embryogenic cultures and regeneration of transformed coniferous plants.